key: cord-337304-2ad2m317
authors: Chang, Le; Zhao, Lei; Gong, Huafei; Wang, Lunan; Wang, Lan
title: Severe Acute Respiratory Syndrome Coronavirus 2 RNA Detected in Blood Donations
date: 2020-07-17
journal: Emerg Infect Dis
DOI: 10.3201/eid2607.200839
sha: 
doc_id: 337304
cord_uid: 2ad2m317

Because of high rates of 2019 novel coronavirus disease in Wuhan, China, Wuhan Blood Center began screening for severe acute respiratory syndrome coronavirus 2 RNA on January 25, 2020. We screened donations in real-time and retrospectively and found plasma samples positive for viral RNA from 4 asymptomatic donors.

Lei Zhao, 1 1) and detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in plasma (2, 3) , the safety of China's blood supply became a major concern (4). Most blood centers and blood banks in China began taking measures to ensure blood safety (5); on January 25, 2020, we began screening all donations collected at the Wuhan Blood Center.

We performed real-time reverse transcription PCR (RT-PCR) for SARS-CoV-2 RNA by using Mul-tiScreen Pro RT-PCR assay (SYM-BIO LifeScience, https://www.sym-bio.com.cn). We performed pool testing by mixing plasma from 6-8 samples or individual testing by using 1.6 mL of plasma. We eluted 100 µL of nucleic acid template and added 40 µL of it to the RT-PCR mix.

By March 4, we had screened 2,430 donations in real-time, including 1,656 platelet and 774 whole blood donations. We identified the first positive donor in our center in a positive pool with a weak amplification of the open reading frame 1ab gene. The donor gave 2 units of platelets on January 28, which were included in the pool. However, the donor's prior donations collected on December 12 and 26 and January 13 were negative for viral RNA. Hubei Province Center for Disease Control and Prevention performed follow-up 

tests on plasma on February 2, which showed a weak positive result near the limit of detection; a throat swab specimen collected from the donor on February 10 also was positive, indicating an extremely low viral load in plasma. The donor reported no symptoms and was quarantined in a cabin hospital in Wuhan until 2 consecutive negative throat swab results on February 23 and February 25 ( Figure) . We also performed retrospective testing of 4,995 donations collected during December 21, 2019-January 22, 2020, by using retained nucleic acid template after routine pool testing. On February 10, we found a positive result in a nucleic acid template derived from donations collected on January 19. We individually tested samples that were in storage at 2°C-8°C for 23 days because no plasma samples stored at -20°C were available. We identified another positive donor of whole blood. We tested plasma products from his donation twice and noted similar results, which suggests that viral RNA is relatively stable in plasma (Appendix Table, https://wwwnc.cdc.gov/EID/article/26/7/20-0839-App1.pdf). We immediately traced all blood products produced from donor 2's whole blood, and they had not been used. Telephone follow-ups on February 15 and 25 showed donor 2 remained asymptomatic and quarantined at home.

In telephone follow-ups with donors who gave blood during January and February, we identified 33 donors who developed a fever after donation; all of their donations were removed from circulation. We performed retrospective individual screening on frozen plasma products from 17 donors and tested the retained nucleic acid templates after routine pool testing of the other 16 donors. We found 2 more positive donors who donated whole blood on January 20. Both had weak positive results, and donors reported fever onset on January 21 ( Figure; Appendix Table) . Donor 3 treated patients infected with SARS-CoV-2 in a Wuhan hospital. His temperature returned to normal 8 days after donation. Donor 4's temperature also returned to normal 7 days after taking self-prescribed antipyretic medications.

By March 4, we identified 4 blood donors in Wuhan whose plasma samples tested positive for SARS-CoV-2 RNA (Figure; Appendix Table) . Samples from these donors were further tested for specific IgG and IgM against SARS-CoV-2 by ELISA; results were negative, indicating the possibility of infection in the early stage and the need to followup with these donors.

We found SARS-CoV-2 RNA in plasma during routine screening of blood donors, considered a healthy population. We tested the 4 donors multiple times, using different sample sources, including sample tubes, retained nucleic acid templates, or blood products, indicating the accuracy and validity of our results (Appendix Table) . One limitation of our study is that we did not have more detailed information on donors 2, 3, and 4. Although we could not confirm virions in blood or whether the virus could be transmitted in blood products, the potential risk should not be neglected. However, detectable RNA might not signify infectivity. Further studies, such as virus culture, should be done to explore the possibility of viremia and follow-up of donors also is essential.

Of note, the donors all donated in late January, and we did not detect SARS-CoV-2 in plasma samples after then, indicating the strict containment measures taken by the government of China were effective. In China, donors are screened for related symptoms and asked if they feel healthy when they donate blood. Having donors call the blood donation center if they have any symptoms after donating is essential to avoid the risk of donation during the COVID-19 incubation period. Moreover, as more asymptomatic cases occur, screening donors for viral RNA with high-sensitivity assays, as we are doing in Hubei Province, will be critical to ensure blood safety. D etection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) typically relies on molecular testing of respiratory tract specimens, although viral RNA can be detected in other specimens (1) . Real-time reverse transcription PCR (rRT-PCR) protocols have been described for SARS-CoV-2, but most involve testing with multiple, singleplex reactions (2) (3) (4) (5) (6) . Such algorithms use large volumes of reagents and limit laboratory testing capacity, both of which have become crucial during the ongoing coronavirus disease pandemic (7). Multiplex assays are commercially available (8,9) but require specific platforms and are more expensive than laboratorydeveloped methods.

Our objective was to develop an internally controlled, triplex assay to detect SARS-CoV-2 RNA in clinical samples. We initially evaluated 6 individual rRT-PCRs, 3 published by the US Centers for Disease Control and Prevention (2) that target the nucleocapsid (N) gene, N1, N2, and N3; and 3 published by Corman, et al. (4) that target RNA-dependent RNA polymerase (RdRp), envelope (E), and N genes. We performed assays in 20 µL reactions of the Luna Universal Probe One-Step RT-qPCR Kit (New England Biolabs, https://www.neb.com) on a Rotor-Gene Q (QIAGEN, https://www.qiagen.com) by using 5 µL of eluate and our standard cycling protocol (10). We extracted total nucleic acids from samples on an Most reverse transcription PCR protocols for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) include 2-3 targets for detection. We developed a triplex, real-time reverse transcription PCR for SARS-CoV-2 that maintained clinical performance compared with singleplex assays. This protocol could streamline detection and decrease reagent use during current high SARS-CoV-2 testing demands.

The Novel Coronavirus Pneumonia Emergency Response Epidemiology Team. Vital surveillances: the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (COVID-19)-China

Clinical features of patients infected with 2019 novel coronavirus in Wuhan

Molecular and serological investigation of 2019-nCoV infected patients: implication of multiple shedding routes

Coronavirus disease 2019: coronaviruses and blood safety

Chinese Society of Blood Transfusion. Recommendations on blood collection and supply during the epidemic of novel coronavirus pneumonia in China

Baofengyi Rd, Wuhan 430030, China; email: wlan66@126.com Triplex Real-Time RT-PCR for Severe Acute Respiratory Syndrome Coronavirus 2